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Purpose: Patients with pancreatic ductal adenocarcinoma (PDAC) who undergo surgical resection and adjuvant chemotherapy have an expected survival of only 2 years due to disease recurrence, frequently in the liver. We investigated the role of liver macrophages in progression of PDAC micrometastases to identify adjuvant treatment strategies that could prolong survival.Experimental Design: A murine splenic injection model of hepatic micrometastatic PDAC was used with five patient-derived PDAC tumors. The impact of liver macrophages on tumor growth was assessed by (i) depleting mouse macrophages in nude mice with liposomal clodronate injection, and (ii) injecting tumor cells into nude versus NOD-scid-gamma mice. Immunohistochemistry and flow cytometry were used to measure CD47 (“don't eat me signal”) expression on tumor cells and characterize macrophages in the tumor microenvironment. In vitro engulfment assays and mouse experiments were performed with CD47-blocking antibodies to assess macrophage engulfment of tumor cells, progression of micrometastases in the liver and mouse survival.Results: In vivo clodronate depletion experiments and NOD-scid-gamma mouse experiments demonstrated that liver macrophages suppress the progression of PDAC micrometastases. Five patient-derived PDAC cell lines expressed variable levels of CD47. In in vitro engulfment assays, CD47-blocking antibodies increased the efficiency of PDAC cell clearance by macrophages in a manner which correlated with CD47 receptor surface density. Treatment of mice with CD47-blocking antibodies resulted in increased time-to-progression of metastatic tumors and prolonged survival.Conclusions: These findings suggest that following surgical resection of PDAC, adjuvant immunotherapy with anti-CD47 antibody could lead to substantially improved outcomes for patients. Clin Cancer Res; 24(6); 1415–25. ©2017 AACR.

Various forms of cell death have been identified, and billions of cells die during development and daily in adult organisms. Clearing dead cells and associated cellular debris is an integral part of tissue homeostasis. While diverse types of phagocytes remove various forms of dying cells during acute kidney injury (AKI), it remains unknown whether boosting removal of a specific form of dying cell would provide a benefit and which cell type should be targeted for phagocytosis-mediated therapy. As there is a lack of viable strategies for the prevention and treatment of AKI, novel therapies and innovative approaches are required. There is a strong demand on developing and analyzing novel models to boost, monitor, and stop phagocytosis of dying cells.

Caspase-dependent apoptosis accounts for approximately 90% of homeostatic cell turnover in the body1, and regulates inflammation, cell proliferation, and tissue regeneration2–4. How apoptotic cells mediate such diverse effects is not fully understood. Here we profiled the apoptotic metabolite secretome and determined its effects on the tissue neighbourhood. We show that apoptotic lymphocytes and macrophages release specific metabolites, while retaining their membrane integrity. A subset of these metabolites is also shared across different primary cells and cell lines after the induction of apoptosis by different stimuli. Mechanistically, the apoptotic metabolite secretome is not simply due to passive emptying of cellular contents and instead is a regulated process. Caspase-mediated opening of pannexin 1 channels at the plasma membrane facilitated the release of a select subset of metabolites. In addition, certain metabolic pathways continued to remain active during apoptosis, with the release of only select metabolites from a given pathway. Functionally, the apoptotic metabolite secretome induced specific gene programs in healthy neighbouring cells, including suppression of inflammation, cell proliferation, and wound healing. Furthermore, a cocktail of apoptotic metabolites reduced disease severity in mouse models of inflammatory arthritis and lung-graft rejection. These data advance the concept that apoptotic cells are not inert cells waiting for removal, but instead release metabolites as ‘good-bye’ signals to actively modulate outcomes in tissues. Apoptotic cells communicate with neighbouring cells by the regulated release of specific metabolites, and a cocktail of select apoptotic metabolites reduces disease severity in mouse models of inflammatory arthritis and lung transplant rejection.

Apoptotic cell clearance (efferocytosis) elicits an anti-inflammatory response by phagocytes, but the mechanisms that underlie this response are still being defined. Here, we uncover a chloride-sensing signalling pathway that controls both the phagocyte 'appetite' and its anti-inflammatory response. Efferocytosis transcriptionally altered the genes that encode the solute carrier (SLC) proteins SLC12A2 and SLC12A4. Interfering with SLC12A2 expression or function resulted in a significant increase in apoptotic corpse uptake per phagocyte, whereas the loss of SLC12A4 inhibited corpse uptake. In SLC12A2-deficient phagocytes, the canonical anti-inflammatory program was replaced by pro-inflammatory and oxidative-stress-associated gene programs. This 'switch' to pro-inflammatory sensing of apoptotic cells resulted from the disruption of the chloride-sensing pathway (and not due to corpse overload or poor degradation), including the chloride-sensing kinases WNK1, OSR1 and SPAK-which function upstream of SLC12A2-had a similar effect on efferocytosis. Collectively, the WNK1-OSR1-SPAK-SLC12A2/SLC12A4 chloride-sensing pathway and chloride flux in phagocytes are key modifiers of the manner in which phagocytes interpret the engulfed apoptotic corpse.

Our bodies turn over billions of cells daily via apoptosis and are in turn cleared by phagocytes via the process of "efferocytosis." Defects in efferocytosis are now linked to various inflammatory diseases. Here, we designed a strategy to boost efferocytosis, denoted "chimeric receptor for efferocytosis" (CHEF). We fused a specific signaling domain within the cytoplasmic adapter protein ELMO1 to the extracellular phosphatidylserine recognition domains of the efferocytic receptors BAI1 or TIM4, generating BELMO and TELMO, respectively. CHEF-expressing phagocytes display a striking increase in efferocytosis. In mouse models of inflammation, BELMO expression attenuates colitis, hepatotoxicity, and nephrotoxicity. In mechanistic studies, BELMO increases ER-resident enzymes and chaperones to overcome protein-folding-associated toxicity, which was further validated in a model of ER-stress-induced renal ischemia-reperfusion injury. Finally, TELMO introduction after onset of kidney injury significantly reduced fibrosis. Collectively, these data advance a concept of chimeric efferocytic receptors to boost efferocytosis and dampen inflammation.

Fertilization is essential for species survival. Although Izumo1 and Juno are critical for initial interaction between gametes, additional molecules necessary for sperm:egg fusion on both the sperm and the oocyte remain to be defined. Here, we show that phosphatidylserine (PtdSer) is exposed on the head region of viable and motile sperm, with PtdSer exposure progressively increasing during sperm transit through the epididymis. Functionally, masking phosphatidylserine on sperm via three different approaches inhibits fertilization. On the oocyte, phosphatidylserine recognition receptors BAI1, CD36, Tim-4, and Mer-TK contribute to fertilization. Further, oocytes lacking the cytoplasmic ELMO1, or functional disruption of RAC1 (both of which signal downstream of BAI1/BAI3), also affect sperm entry into oocytes. Intriguingly, mammalian sperm could fuse with skeletal myoblasts, requiring PtdSer on sperm and BAI1/3, ELMO2, RAC1 in myoblasts. Collectively, these data identify phosphatidylserine on viable sperm and PtdSer recognition receptors on oocytes as key players in sperm:egg fusion., Izumo and Juno are receptors on sperm and eggs respectively required for fusion, but other factors for sperm-egg fusion are poorly studied. Here, the authors report that phosphatidylserine, found mainly on cells marked for death, is also present on motile sperm, recognized by egg receptors, and is required for sperm-egg fusion.

Development and routine tissue homeostasis require a high turnover of apoptotic cells. These cells are removed by professional and non-professional phagocytes via efferocytosis1. How a phagocyte maintains its homeostasis while coordinating corpse uptake, processing ingested materials and secreting anti-inflammatory mediators is incompletely understood1,2. Here, using RNA sequencing to characterize the transcriptional program of phagocytes actively engulfing apoptotic cells, we identify a genetic signature involving 33 members of the solute carrier (SLC) family of membrane transport proteins, in which expression is specifically modulated during efferocytosis, but not during antibody-mediated phagocytosis. We assessed the functional relevance of these SLCs in efferocytic phagocytes and observed a robust induction of an aerobic glycolysis program, initiated by SLC2A1-mediated glucose uptake, with concurrent suppression of the oxidative phosphorylation program. The different steps of phagocytosis2—that is, ‘smell’ (‘find-me’ signals or sensing factors released by apoptotic cells), ‘taste’ (phagocyte–apoptotic cell contact) and ‘ingestion’ (corpse internalization)—activated distinct and overlapping sets of genes, including several SLC genes, to promote glycolysis. SLC16A1 was upregulated after corpse uptake, increasing the release of lactate, a natural by-product of aerobic glycolysis3. Whereas glycolysis within phagocytes contributed to actin polymerization and the continued uptake of corpses, lactate released via SLC16A1 promoted the establishment of an anti-inflammatory tissue environment. Collectively, these data reveal a SLC program that is activated during efferocytosis, identify a previously unknown reliance on aerobic glycolysis during apoptotic cell uptake and show that glycolytic by-products of efferocytosis can influence surrounding cells. Distinct transcriptional programs are activated during different stages of apoptotic cell engulfment, including a unique program of genes coding for solute carrier proteins and enzymes in the glycolytic pathway.

Anuran tadpoles can regenerate their tails after amputation. However, they occasionally form ectopic limbs instead of the lost tail part after vitamin A treatment. This is regarded as an example of a homeotic transformation. In this phenomenon, the developmental fate of the tail blastema is apparently altered from that of a tail to that of limbs, indicating a realignment of positional information in the blastema. Morphological observations and analyses of the development of skeletal elements during the process suggest that positional information in the blastema is rewritten from tail to trunk specification under the influence of vitamin A, resulting in limb formation. Despite the extensive information gained from morphological observations, a comprehensive understanding of this phenomenon also requires molecular data. We review previous studies related to anuran homeotic transformation. The findings of these studies provide a basis for evaluating major hypotheses and identifying molecular data that should be prioritized in future studies. Finally, we argue that positional information for the tail blastema changes to that for a part of the trunk, leading to homeotic transformations. To suggest this hypothesis, we present published data that favor the rewriting of positional information.

Purpose: Patients with pancreatic ductal adenocarcinoma (PDAC) who undergo surgical resection and adjuvant chemotherapy have an expected survival of only 2 years due to disease recurrence, frequently in the liver. We investigated the role of liver macrophages in progression of PDAC micrometastases to identify adjuvant treatment strategies that could prolong survival. Experimental Design: A murine splenic injection model of hepatic micrometastatic PDAC was used with five patient-derived PDAC tumors. The impact of liver macrophages on tumor growth was assessed by (i) depleting mouse macrophages in nude mice with liposomal clodronate injection, and (ii) injecting tumor cells into nude versus NOD-scid-gamma mice. Immunohistochemistry and flow cytometry were used to measure CD47 (“don't eat me signal”) expression on tumor cells and characterize macrophages in the tumor microenvironment. In vitro engulfment assays and mouse experiments were performed with CD47-blocking antibodies to assess macrophage engulfment of tumor cells, progression of micrometastases in the liver and mouse survival. Results: In vivo clodronate depletion experiments and NOD-scid-gamma mouse experiments demonstrated that liver macrophages suppress the progression of PDAC micrometastases. Five patient-derived PDAC cell lines expressed variable levels of CD47. In in vitro engulfment assays, CD47-blocking antibodies increased the efficiency of PDAC cell clearance by macrophages in a manner which correlated with CD47 receptor surface density. Treatment of mice with CD47-blocking antibodies resulted in increased time-to-progression of metastatic tumors and prolonged survival. Conclusions: These findings suggest that following surgical resection of PDAC, adjuvant immunotherapy with anti-CD47 antibody could lead to substantially improved outcomes for patients. Clin Cancer Res; 24(6); 1415–25. ©2017 AACR.

Nearly every tissue in the body undergoes routine turnover of cells as part of normal healthy living. The majority of these cells undergoing turnover die via apoptosis, and then are rapidly removed by phagocytes by the process of efferocytosis that is anti-inflammatory. However, a number of pathologies have recently been linked to defective clearance of apoptotic cells. Perturbed clearance arises for many reasons, including overwhelming of the clearance machinery, disruptions at different stages of efferocytosis, and/or responses of phagocytes during efferocytosis, all of which can alter the homeostatic tissue environment. This review covers linkages of molecules involved in the different phases of efferocytosis to disease pathologies that can arise due to their loss or altered function.

Sterol-regulatory element-binding proteins (SREBPs) are key transcription factors regulating cholesterol and fatty acid biosynthesis. SREBP activity is tightly regulated to maintain lipid homeostasis, and is modulated upon extracellular stimuli such as growth factors. While the homeostatic SREBP regulation is well studied, stimuli-dependent regulatory mechanisms are still elusive. Here we demonstrate that SREBPs are regulated by a previously uncharacterized mechanism through transforming growth factor-β activated kinase 1 (TAK1), a signaling molecule of inflammation. We found that TAK1 binds to and inhibits mature forms of SREBPs. In an in vivo setting, hepatocyte-specific Tak1 deletion upregulates liver lipid deposition and lipogenic enzymes in the mouse model. Furthermore, hepatic Tak1 deficiency causes steatosis pathologies including elevated blood triglyceride and cholesterol levels, which are established risk factors for the development of hepatocellular carcinoma (HCC) and are indeed correlated with Tak1-deficiency-induced HCC development. Pharmacological inhibition of SREBPs alleviated the steatosis and reduced the expression level of the HCC marker gene in the Tak1-deficient liver. Thus, TAK1 regulation of SREBP critically contributes to the maintenance of liver homeostasis to prevent steatosis, which is a potentially important mechanism to prevent HCC development.

Programmed cell death, a physiologic process for removing cells, is critically important in normal development and for elimination of damaged cells. Conversely, unattended cell death contributes to a variety of human disease pathogenesis. Thus, precise understanding of molecular mechanisms underlying control of cell death is important and relevant to public health. Recent studies emphasize that transforming growth factor-β-activated kinase 1 (TAK1) is a central regulator of cell death and is activated through a diverse set of intra- and extracellular stimuli. The physiologic importance of TAK1 and TAK1-binding proteins in cell survival and death has been demonstrated using a number of genetically engineered mice. These studies uncover an indispensable role of TAK1 and its binding proteins for maintenance of cell viability and tissue homeostasis in a variety of organs. TAK1 is known to control cell viability and inflammation through activating downstream effectors such as NF-κB and mitogen-activated protein kinases (MAPKs). It is also emerging that TAK1 regulates cell survival not solely through NF-κB but also through NF-κB-independent pathways such as oxidative stress and receptor-interacting protein kinase 1 (RIPK1) kinase activity-dependent pathway. Moreover, recent studies have identified TAK1's seemingly paradoxical role to induce programmed necrosis, also referred to as necroptosis. This review summarizes the consequences of TAK1 deficiency in different cell and tissue types from the perspective of cell death and also focuses on the mechanism by which TAK1 complex inhibits or promotes programmed cell death. This review serves to synthesize our current understanding of TAK1 in cell survival and death to identify promising directions for future research and TAK1's potential relevance to human disease pathogenesis.

Activation of the TAK1 kinase drives RIPK3-dependent necrosis and inhibits apoptosis downstream of TNF-α stimulation., TNF activates three distinct intracellular signaling cascades leading to cell survival, caspase-8–mediated apoptosis, or receptor interacting protein kinase 3 (RIPK3)–dependent necrosis, also called necroptosis. Depending on the cellular context, one of these pathways is activated upon TNF challenge. When caspase-8 is activated, it drives the apoptosis cascade and blocks RIPK3-dependent necrosis. Here we report the biological event switching to activate necrosis over apoptosis. TAK1 kinase is normally transiently activated upon TNF stimulation. We found that prolonged and hyperactivation of TAK1 induced phosphorylation and activation of RIPK3, leading to necrosis without caspase activation. In addition, we also demonstrated that activation of RIPK1 and RIPK3 promoted TAK1 activation, suggesting a positive feedforward loop of RIPK1, RIPK3, and TAK1. Conversely, ablation of TAK1 caused caspase-dependent apoptosis, in which Ripk3 deletion did not block cell death either in vivo or in vitro. Our results reveal that TAK1 activation drives RIPK3-dependent necrosis and inhibits apoptosis. TAK1 acts as a switch between apoptosis and necrosis.

Hematopoietic cell survival and death is critical for development of a functional immune system. Here, we report that a protein kinase, TAK1, is selectively required for resident macrophage integrity during embryogenesis. Hematopoietic lineage-specific deletion of Tak1 gene (Tak1HKO) caused accumulation of cellular debris in the thymus in perinatal mice. Although no overt alteration in thymocytes and blood myeloid populations was observed in Tak1HKO mice, we found that thymic and lung macrophages were diminished. In the in vitro setting, Tak1 deficiency caused profound disruption of lysosomes and killed bone marrow-derived macrophages (BMDMs) without any exogenous stressors. Inhibition of the lysosomal protease, cathepsin B, partially blocked Tak1-deficient BMDM death, suggesting that leakage of the lysosomal contents is in part the cause of cell death. To identify the trigger of this cell death, we examined involvement of TNF and Toll-like receptor pathways. Among them, we found that deletion of Tnfr1 partially rescued cell death. Finally, we show that Tnfr1 deletion partially restored thymic and lung macrophages in vivo. These results suggest that autocrine and potentially paracrine TNF kills Tak1-deficient macrophages during development. Our results reveal that TAK1 signaling maintains proper macrophage populations through protecting lysosomal integrity.

Sustained endoplasmic reticulum (ER) stress disrupts normal cellular homeostasis and leads to the development of many types of human diseases, including metabolic disorders. TAK1 (also known as MAP3K7) is a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family and is activated by a diverse set of inflammatory stimuli. Here, we demonstrate that TAK1 regulates ER stress and metabolic signaling through modulation of lipid biogenesis. We found that deletion of Tak1 increased ER volume and facilitated ER-stress tolerance in cultured cells, which was mediated by upregulation of sterol-regulatory-element-binding protein (SREBP)-dependent lipogenesis. In the in vivo setting, central nervous system (CNS)-specific Tak1 deletion upregulated SREBP-target lipogenic genes and blocked ER stress in the hypothalamus. Furthermore, CNS-specific Tak1 deletion prevented ER-stress-induced hypothalamic leptin resistance and hyperphagic obesity under a high-fat diet (HFD). Thus, TAK1 is a crucial regulator of ER stress in vivo, which could be a target for alleviation of ER stress and its associated disease conditions.

Sustained endoplasmic reticulum (ER) stress disrupts normal cellular homeostasis and leads to the development of many types of human diseases, including metabolic disorders. TAK1 (also known as MAP3K7) is a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family and is activated by a diverse set of inflammatory stimuli. Here, we demonstrate that TAK1 regulates ER stress and metabolic signaling through modulation of lipid biogenesis. We found that deletion of Tak1 increased ER volume and facilitated ER-stress tolerance in cultured cells, which was mediated by upregulation of sterol-regulatory-element-binding protein (SREBP)-dependent lipogenesis. In the in vivo setting, central nervous system (CNS)-specific Tak1 deletion upregulated SREBP-target lipogenic genes and blocked ER stress in the hypothalamus. Furthermore, CNS-specific Tak1 deletion prevented ER-stress-induced hypothalamic leptin resistance and hyperphagic obesity under a high-fat diet (HFD). Thus, TAK1 is a crucial regulator of ER stress in vivo, which could be a target for alleviation of ER stress and its associated disease conditions.

Macrophages play diverse roles in tissue homeostasis and immunity, and canonically activated macrophages are critically associated with acute inflammatory responses. It is known that activated macrophages undergo cell death after transient activation in some settings, and the viability of macrophages impacts on inflammatory status. Here we report that TGFβ- activated kinase (TAK1) activators, TAK1-binding protein 1 (TAB1) and TAK1-binding protein 2 (TAB2), are critical molecules in the regulation of activated macrophage survival. While deletion of Tak1 induced cell death in bone marrow derived macrophages even without activation, Tab1 or Tab2 deletion alone did not profoundly affect survival of naive macrophages. However, in lipopolysaccharide (LPS)-activated macrophages, even single deletion of Tab1 or Tab2 resulted in macrophage death with both necrotic and apoptotic features. We show that TAB1 and TAB2 were redundantly involved in LPS-induced TAK1 activation in macrophages. These results demonstrate that TAK1 activity is the key to activated macrophage survival. Finally, in an in vivo setting, Tab1 deficiency impaired increase of peritoneal macrophages upon LPS challenge, suggesting that TAK1 complex regulation of macrophages may participate in in vivo macrophage homeostasis. Our results demonstrate that TAB1 and TAB2 are required for activated macrophages, making TAB1 and TAB2 effective targets to control inflammation by modulating macrophage survival.

TGF-β activated kinase 1 (TAK1) is a mediator of various cytokine signaling pathways. Germline deficiency of Tak1 causes multiple abnormalities, including dilated blood vessels at midgestation. However, the mechanisms by which TAK1 regulates vessel formation have not been elucidated. TAK1 binding proteins 1 and 2 (TAB1 and TAB2) are activators of TAK1, but their roles in embryonic TAK1 signaling have not been determined. In the present study, we characterized mouse embryos harboring endothelial-specific deletions of Tak1, Tab1, or Tab2 and found that endothelial TAK1 and TAB2, but not TAB1, were critically involved in vascular formation. TAK1 deficiency in endothelial cells caused increased cell death and vessel regression at embryonic day 10.5 (E10.5). Deletion of TNF signaling largely rescued endothelial cell death in TAK1-deficient embryos at E10.5. However, embryos deficient in both TAK1 and TNF signaling still exhibited dilated capillary networks at E12.5. TAB2 deficiency caused reduced TAK1 activity, resulting in abnormal capillary blood vessels, similar to the compound deficiency of TAK1 and TNF signaling. Ablation of either TAK1 or TAB2 impaired cell migration and tube formation. Our results show that endothelial TAK1 signaling is important for 2 biologic processes in angiogenesis: inhibiting TNF-dependent endothelial cell death and promoting TNF-independent angiogenic cell migration.

Mice with a keratinocyte-specific deletion of Tak1 exhibit severe skin inflammation due to hypersensitivity to tumor necrosis factor (TNF) killing. Here we have examined the mechanisms underlying this hypersensitivity. We found that TAK1 deficiency up-regulates reactive oxygen species (ROS) resulting in cell death upon TNF or oxidative stress challenge. Because blockade of NF-κB did not increase ROS or did not sensitize cells to oxidative stress in keratinocytes TAK1 regulates ROS mainly through the mechanisms other than those mediated by NF-κB. We found that c-Jun was decreased in TAK1-deficient keratinocytes and that ectopic expression of c-Jun could partially inhibit TNF-induced increase of ROS and cell death. Finally, we show that, in an in vivo setting, the antioxidant treatment could reduce an inflammatory condition in keratinocyte-specific Tak1 deletion mice. Thus, TAK1 regulates ROS partially through c-Jun, which is important for preventing ROS-induced skin inflammation.

The liver is the first line of defense from environmental stressors in that hepatocytes respond to and metabolize them. Hence, hepatocytes can be damaged by stressors. Protection against hepatic cell damage and cell death is important for liver function and homeostasis. TAK1 (MAP3K7) is an intermediate of stressors such as bacterial moieties-induced signal transduction pathways in several cell types. Tak1 deficiency has been reported to induce spontaneous hepatocellular carcinoma. However, the regulatory mechanism of TAK1 activity in liver stress response has not yet been defined. Here we report that activation of TAK1 through TAK1 binding protein 2 (TAB2) is required for liver protection from stressors. We found that a bacterial moiety, lipopolysaccharides (LPS), activated TAK1 in primary hepatocytes, which was diminished by deletion of TAB2. Mice having hepatocyte-specific deletion of the Tab2 gene exhibited only late-onset moderate liver lesions but were hypersensitive to LPS-induced liver injury. Furthermore, we show that a chemical stressor induced greatly exaggerated liver injury in hepatocyte-specific Tab2-deficient mice. These results demonstrate that TAB2 is a sensor of stress conditions in the liver and functions to protect the liver by activating the TAK1 pathway.